PROJECT SUMMARY/ABSTRACT Pathologic dysregulation of the coagulation system is a major contributor to human morbidity and mortality, resulting in either excessive bleeding or clotting. Significant progress has been made in the identification of genetic regulators of the coagulation cascade, but many unknown modifier genes contribute to the variable disease severity and penetrance observed among patients and families with hemostatic and thrombotic disorders. Understanding such modifiers could help classify patients at higher risk for pathology as well as identify novel therapeutic targets. Although major improvements to treatment of hemorrhagic and bleeding disorders have been made with blood products and human derived or recombinant coagulation factors, these have limited shelf life and storage conditions, and require intravenous infusion. Anticoagulation has seen a surge in recent years with many new direct acting oral anticoagulants, but their mechanisms of action are limited to the coagulation cascade. Building on our previous work, this project will take advantage of powerful genetic tools, including genome editing nucleases, next generation sequencing, and the zebrafish. We will conduct a large scale interrogation of the genome to discover hemostasis regulatory genes with the potential to modify the severity of human coagulation disorders. We have developed a panel of clotting factor mutant zebrafish using robust genome editing nucleases (TALENS and CRISPR/Cas) and conducted chemical mutagenesis experiments that have identified potential suppressor mutant lines harboring prospective thrombosis and hemostasis modifier genes. This panel of mutant fish will also be used for unbiased assays to identify novel lead molecules that suppress hemorrhage or thrombosis. The approaches described in this proposal will lead to the identification of the key non-canonical factors regulating hemostasis and thrombopoiesis, some of which will likely prove to be important genetic modifying factors in humans, or will suggest novel species-specific but biologically insightful regulatory mechanisms. This will shed light on the regulatory mechanisms of hemostasis, and the modifiers will also be candidate diagnostic and therapeutic targets for human thrombotic and hemorrhagic diseases. These targets will be utilized to develop potential innovative agents and new therapeutic classes for treatment of hemorrhage and thrombosis that could benefit the general population, as well as patients with bleeding and thrombotic disorders.